Archaesomes, archaeosomes containing coenzyme Q10 and other types of liposomes containing coenzyme Q10 adjuvants and as delivery vehicles

ABSTRACT

Novel archaeosome compositions and their use in vaccine formulations as adjuvants and/or delivery systems, to enhance the immune response to immunogens in an animal such as a human, are described. Another aspect relates to the use of these archaeosomes to enhance the delivery of compounds such as pharmaceuticals to specific cell types and tissues in animals and other life forms, via various routes of administration such as subcutaneous, intramuscular, and oral. The efficacy of the archaeosomes and also of conventional liposomes can be further improved in these applications, by incorporation of coenzyme Q 10  and/or polyethyleneglycol-lipid conjugate into liposomes made from these archaeosomes.

This application is a division of Ser. No. 09/077,956 filed Jun. 12,1998, which was based on PCT/CA96/00835 filed Dec. 13, 1996 now U.S.Pat. No. 6,132,789 which claims benefit over No. 60/008,724 filed Dec.15, 1995.

FIELD OF THE INVENTION

This invention relates to liposomes (closed lipid vesicles) made fromarchaeobacterial lipids, from non-archaeobacterial lipids, and mixturesthereof, and to the use of such liposomes for the enhanced delivery ofpharmaceutical and other compounds to specific cell types such asmacrophages/phagotocytes/antigen processing cells and to specifictissues in life-forms such as humans, and for the enhancement of theimmune response to antigen(s) presented to a life-form such as a human.The vesicles of this invention may be used in vaccine formulations afterencapsulation of, or in conjunction with, one or more immunogen, with orwithout mediation by the presence of other adjuvants or compounds. Theinvention may also be used, without limitations, for delivery of drugs,antibiotics, pharmaceuticals, biological compounds such as enzymes orDNA or hormones, therapeutics, imaging agents etc to specific cell typesor specific tissues in an animal such as a human and other life-forms.Another application may be to use antibiotics/antiviral agentsencapsulated in archaeosomes to treat diseases, where the infectiveorganisms may reside as intracellular reservoirs (such as inmacrophages) for re-infection.

DESCRIPTION OF THE PRIOR ART

Liposomes are closed lipid vesicles containing an entrapped aqueousvolume. The hydrophilic head groups of the lipids forming liposomes areoriented towards the aqueous environments present inside and outside theliposomes, whereas the hydrophobic regions of the lipids are sandwichedbetween the polar head groups and away from the aqueous environments.Liposomes may be unilamellar containing a single lipid bilayer, ormultilamellar containing multiple bilayers (onion-like in structure)with an aqueous space separating each bilayer from the other. Varioustechniques for forming liposomes have been described in the literature,including but not limited to, pressure extrusion, detergent dialysis,dehydration-rehydration, reverse-phase evaporation, remote loading,sonication and other methods (13). Liposomes made from conventionalester phospholipids such as phosphatidylcholine are referred to hereinas conventional liposomes, even if they contain sterols or othercompounds in their bilayer.

Liposomes consisting of a lipid bilayer, a monolayer or a combinationthereof, made from any lipid(s) which include in their composition etherlipids extracted from or found in Archaeobacteria, or those synthesizedto mimic lipids found in archaeobacteria, are referred to herein asarchaeosomes.

Archaea (Archaeobacteria) are considered to be distinct from eubacteriaand eukaryotes, and they include aerobic, anaerobic, thermophilic,extremely thermophilic, thermoacidophilic, and halophilicmicroorganisms. Total lipid extracts from individual species of Archaeaconsist of polar ether lipids and from 5 to 20% neutral lipids. Thepolar ether lipids of Archaea consist of branched phytanyl chains whichare usually saturated and are attached via ether bonds to the glycerolcarbons at the sn-2,3 positions (8). In contrast to this, inconventional phospholipids found in Eubacteria and Eukaryotes, fattyacyl chains which may be unsaturated, are attached via ester bonds tothe sn-1,2 carbons of the glycerol backbone. The core structures of thearchaeobacterial ether lipids (the polar head groups removed byhydrolysis) consist of the standard diether lipid(2,3-di-O-phytanyl-sn-glycerol or archaeol), and the standard tetraetherlipid (2,2′,3,3′-tetra-O-dibiphytanyl-sn-diglycerol or caldarchaeol) andmodifications thereof (8). Diether lipids are monopolar like theconventional phospholipids, whereas the tetraether lipids are bipolar.The polar head groups, attached to the sn-1 glycerol carbon in thediethers and to the sn-1 and sn-1′ glycerol carbons in the tetraethers,can vary and may include phospho groups, glyco groups, phosphoglycogroups, polyol groups, c hydroxyl groups (18). In contrast to thephosphatidylcholine conventional lipid commonly used in liposomeformulations, the phosphocholine head group is very rarely found inarchaeobacterial polar lipids. Archaea provide a large selection oflipids to screen for the preparation of vesicles having the propertiesuseful for specific applications, and overcome the difficultiesassociated with conventional lipids such as low adjuvanticity andinstability.

There is much interest in the use of liposomes for medical,pharmaceutical, and other commercial applications. Most of the researchreported on liposomes to-date, has been conducted using conventionalphospholipids sometimes mixed with sterols (e.g., cholesterol) or othercompounds to improve stability, rather than using eitherarchaeobacterial or non-archaeobacterial ether lipids.

In a comparative study on the uptake of liposomes made with1,2-diacyl-sn-glycero-3-phosphocholine and its ether analog, by culturedrat liver hepatocytes, the cellular uptake of both liposome types wasfound to be similar (21). In another study, liposomes made with eitherdipalmitoyl phosphatidylcholine or its ether analogue1-O-octadecyl-2-O-methyl-rac-glycerol-3-phosphocholine, werephagocytosed at about the same rate by J774.E1 macrophage cells (6).Therefore, from these disclosures it would be expected that liposomesmade with ether lipids, including by extension those from ether lipidseither extracted from Arcaea or from ether lipids chemically synthesizedto mimic the unique lipid structures of Archaea, would be taken up bycertain cells such as macrophages, to a similar extent as conventionalliposomes. However, the current invention proves to the contrary,showing enhanced phagocytosis of vesicles (archaeosomes) made witharchaeobacterial ether lipids.

Intracellular delivery of antibiotics and other drugs to controlpathogens which reside within certain cell types such as macrophages isa current problem, e.g., the bacterium Mycobacterium tuberculosis whichcauses tuberculosis, viruses such as the human immunodeficiency virus(HIV) which causes acquired immune deficiency syndrome (AIDS), andparasites which cause malaria. A superior uptake of archaeosomes madewith ether lipids of Archaea would have commercial utility in enhancingthe delivery of drugs, antigens, and other compounds targeted fordelivery to phagocytic cells of a life-form, such as a human.

There is considerable interest in the potential use of liposomes in thefield of vaccine applications. Liposomes prepared from conventionalphospholipids, sometimes mixed with cholesterol or other compounds(conventional liposomes) have been tested as potential antigencarriers/vehicles. Allison and Gregoriadis (1) reported that liposomesprepared from egg phosphatidylcholine had some adjuvant activity,provided a negatively charged lipid was included in the liposomecomposition. Since conventional liposomes often demonstrate only smalladjuvant effects as compared with administration of the free antigen,various immunostimulatory substances such as lipid A have beenco-incorporated into the liposomes, together with the antigen (4).However, as is the case with lipid A and Freund's adjuvant,immunostimulatory substances may have toxicity associated problems,making them unsuitable for vaccine applications.

The humoral immune response, in mice, to bovine serum albuminencapsulated in liposomes made with dialkyl-ethersn-3-phosphatidylcholine was lower than that obtained with similarliposomes made with diacyl-ester sn-3 phosphatidylcholine (17). There isno teaching in the prior art to suggest true compared with liposomesmade using conventional phospholipids, those made using archaeobacterialether lipids would have a superior adjuvant effect in stimulating theimmune response to an antigen administered into an animal by variousroutes (including but not limited to intramuscular (i.m.), intravenous(i.v.), intraperitoneal (i.p.), subcutaneous (s.c.), and peroral(p.o.)).

Coenzyme Q₁₀ (also known as CoQ₁₀, ubiquinone-10 or ubidecarenone-10) ispresent in mammalian cells having mitochondria where it is a redoxcomponent in the respiratory chain. Since decreased levels of CoQ₁₀ havebeen implicated in certain pathological conditions such as myocardiacinsufficiency, cardiac infarction, muscular dystrophy, arterioushypertension, and in the symptoms of ageing, it has been considered fortherapeutic applications in such cases. Deficiency of CoQ₁₀ has alsobeen reported in AIDS patients (5). CoQ₁₀ is extremely hydrophobic innature and unless its chemical structure is artificially modified, it isnot soluble in aqueous buffers or water. Similarities are noted betweenCoQ₁₀ and vitamin E, with respect to serving as immune stimulators andas anti-oxidants. CoQ₁₀ as an emulsion with detergents has been shown toenhance the in vivo phagocytic activity in animal models (3). LabelledCoQ₁₀ has been used in conventional liposomes as a marker for myocardialimaging and for studying tissue distribution of conventional liposomescoated with polysaccharides (7,22). In liposomes, CoQ₁₀ is associatedwith the lipid layer of the vesicles. However, none of these or otherprior art publications teach that the combination of CoQ₁₀ inarchaeosomes would enhance the phagocytosis of the resultant vesicles bymacrophage cells (compared to that of the archaeosomes without CoQ₁₀),or that such a combination would allow the alteration in targetingprofiles to specific tissues when the vesicles are administered to ananimal via different routes, or that such combinations would furtherenhance the immune response to co-administered immunogen(s) (currentclaimed invention). Similarly, the prior art does not teach that thecombination of CoQ₁₀ in conventional liposomes would increase thephagocytosis of the resultant liposomes by macrophages, or allow for thealteration of tissue targeting profiles when the liposomes areadministered to an animal by different routes, or that liposomal CoQ₁₀combination would enhance the immune response to co-administeredimmunogen(s) compared to the liposomal immunogen in the absence ofCoQ₁₀.

SUMMARY OF THE INVENTION

It is an object of this invention to use archaeosomes as beneficialcarriers of antigens, immunogenic compounds, DNA, drugs, therapeuticcompounds, pharmaceutical compounds, imaging agents or tracers, and todeliver these to specific cells such as the macrophages or to specifictissues, in life-forms such as the human.

It is a further object of this invention to provide archaeosomes thathave enhanced adjuvant activity for the generation of an immune responseto an immunogen, and for vaccine applications in an animal such as ahuman, wherein the antigen(s) can be encapsulated in, can be associatedwith, or not associated with the archaeosomes, at the time ofadministration via different routes. In some instances the immuneresponse (the level of response and its duration) to an immunogendelivered via an archaeosome being comparable to that obtained withFreund's adjuvant as the immunostimulator.

Yet another object of the invention is to use archaeosomes prepared withlipids containing a high proportion of tetraether bipolar lipid(s) thatare found in, or mimic those found in, members of archaeobacteria, toenhance and prolong the immune response to an immunogen that is eithercodministered as part of the archaeosome or administered at the sametime as the archaeosome, into an animal.

It is another object of the invention to incorporate coenzyme Q₁₀ intoarchaeosomes, or into liposomes prepared exclusively from lipids otherthan archaeobacterial-like ether lipids, to enhance the phagocytosis ofthe respective archaeosomes/liposomes, and/or to enhance the delivery ofCoQ₁₀ as well as other associated drug(s), and to enhance the immuneresponse to an antigen associated with the respectivearchaeosomes/liposomes.

It is another object of this invention to incorporate CoQ₁₀ intoarchaeosomes and conventional liposomes, sometimes in combination withpolyethylene glycol lipid conjugates, to increase the delivery ofvarious associated compounds to specific organ tissues when therespective vesicles are administered to an animal via various routessuch as p.o., i.m., i.v., s.c, and i.p. The combination of CoQ₁₀ inarchaeosomal or conventional liposomal vesicles, including vesicles thatmay have been sterically stabilized by association withpolyethyleneglycol conjugates, would therefore further increase theutility of archaeosomes and of conventional liposomes, for delivery ofcompounds, including immunogens and CoQ₁₀ itself, to phagocytic cellsand to specific tissues.

It is yet another object of the invention to incorporate optimal amountsof archaeobacterial ether lipid(s) into mixtures with conventionallipids to prepare vesicles that have the above improved characteristics.

All of the above aspects of the current invention are interrelated inconcept.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Fluorescence micrographs of cells incubated with Methanosarcinamazei archaeosomes, or with conventional liposomes prepared fromDMPC:DMPG:CHOL, each vesicle type containing CF. Panels A₁, B₁, C₁,murine peritoneal macrophages. Panels A₂, B₂, C₂, J774A.1 macrophages.Panels A3, B₃, C₃, HEp-2 cells. Panels A₁₋₃, archaeosomes; Panels B₁₋₃,conventional liposomes; and panels C₁₋₃, cells without added vesicles.The presence of yellow-fluorescent liposomes are indicated in the blackand white photos by light areas. Magnification bar=20 μm.

FIG. 2. Fluorescence micrographs of murine peritoneal macrophages,J774A.1, and HEp-2 cells incubated with multilamellar M. mazeiarchaeosomes (A₁, B₁, C₁), or multilamellar conventional liposomesprepared from DMPC:DMPG:CHOL (A₂, B₂, C₂), each vesicle type containingCF. Panels A₁₋₂, J774A.1 cells. Panels B₁₋₂, murine peritonealmacrophages. Panels C₁₋₂, HEp-2 cells. Yellow-fluorescence of vesiclesappears in the black and white photos as light areas. Magnificationbar=20 μm.

FIG. 3. Tissue distribution of (A) conventional liposomes(DSPC:DCP:CHOL) and, (B) CoQ₁₀-conventional liposomes(DSPC:DCP:CHOL:Q₁₀) 24 hours after oral and parenteral administration tomice. The data shown are ± sample standard error from the mean.

FIG. 4. Tissue distribution of (A) PEG-conventional liposomes(DSPC:DCP:CHOL:DSPE-PEG) and, (B) PEG-CoQ₁₀-conventional liposomes(DSPC:DCP:CHOL:DSPE-PEG:Q₁₀) 24 hours after oral and parenteraladministration to mice. The data shown are ± sample standard error fromthe mean.

FIG. 5. Tissue distribution of (A) archaeosomes (M. mazei TPL) and, (B)CoQ₁₀-archaeosomes (M.mazei TPL:Q₁₀) 24 hours after oral and parenteraladministration to mice. The data shown are ± sample standard error fromthe mean.

FIG. 6. Tissue distribution of (A) PEG-archaeosomes (M.mazeiTPL:DSPE-PEG) and, (B) PEG-CoQ₁₀-archaeosomes (M.mazei TPL:DSPE-PEG:Q₁₀)24 hours after oral and parenteral administration to mice. The datashown are ± sample standard error from the mean.

FIG. 7. Murine humoral responses to Cholera B subunit as antigen. Firstand second injections (i.p.) consisted of: B, 1.0 μg of antigen in PBS(no adjuvant), FA, 1.0 μg of antigen+Freund's adjuvant; M. smithii,archaeosomes (1.21 mg lipid, 1.0 μg antigen encapsulated); and M.smithii+B, bare archaeosomes (1.21 mg lipid) followed 1 hour later by1.0 μg of antigen in PBS. Immunizations were at days 0 and 14.

FIG. 8. Comparison of murine humoral responses to bovine serum albumin(BSA) incorporated into a variety of lipid vesicles/adjuvants. At eachinjection, the mice received 25 μg BSA. Mice were injected i.p. at days0, 10 and 52, and sera collected 4 days after the second and thirdinjections. Archaeosomes were prepared from the TPL extracted from theindicated archaeobacterium. Sera diluted 1:400. Vesicle/adjuvantcomposition (mg lipid/boost): BSA—no adjuvant or vesicle, BSA diluted inPBS; FA-BSA emulsified in Freund's adjuvant; T. acidophilum (0.75 mg);M. mazei (0.64 mg); M. smithii (0.57 mg); M. voltae (1.83 mg); M.hungatei (1.09 mg); M. concilii (0.68 mg); M. stadtmanae (0.51 mg);PC:PG (DMPC:DMPG, 2.11 mg); PC:PG:CHOL (DMPC:DMPG:CHOL, 0.12 mg); andPC:DCP:CHOL (DMPC:DCP:CHOL, 0.87 mg). Data are the means from mice induplicates.

FIG. 9. Relationship between the number of immunizations with BSAencapsulated in archaeosomes and the resultant anti-BSA antibody titresin mouse sera. Each mouse received 25 μg BSA per i.p. injection eitheras bare antigen, encapsulated in M. smithii archaeosomes (0.58 mglipid), or emulsified in FA. Mice were injected at days 0, 24, 54 and108. Sera diluted 1:400. Injection regimen: 0/4, immunized up to 4 timeswith the bare antigen; 1/3, first injection with encapsulated antigenand remaining 3 boosts with the bare antigen; 2/2, first 2 injectionswith encapsulated antigen and remaining 2 boosts with the bare antigen;3/1, first 3 injections with encapsulated antigen and remaining boostwith the bare antigen; 4/0, 4 injections with encapsulated antigen; andFA, first injection with CFA, second with IFA, remaining 2 boosts withthe bare antigen. Data are expressed as the means for duplicate mice.

FIG. 10. Anti-BSA antibody isotypes found in mouse sera followingimmunizations with BSA encapsulated in archaeosomes. Injections weregiven i.m. to mice at days 0 and 14, using 12.5 μg BSA/injection.Archaeosomes carrying 12.5 μg BSA, prepared from the TPL, correspond todry weights/injection of 0.69 mg (M. mazei), 0.55 mg (M. hungatei), and1.5 mg (T. acidophilum). Sera samples were obtained on day 25 post firstinjection. Also shown are isotyping results for injections of bare BSA(BSA) and BSA with Freund's adjuvant (FA).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The Archaea (Archaeobacteria) produce many different polyether lipidstructures that are useful for the production of vesicles (archaeosomes)that have unique properties. Of the available species of Archaea as aclass of organisms, we chose several as illustrative examples, toencompass a broad spectrum of ether lipid structures; namely,Halobacterium cutirubrum, Methanococcus mazei, Methanospirillumhungatei, Methanococcus jannaschii, Methanosphaera stadtmanae,Methanobrevibacter smithii, Methanococcus voltae, Thermoplasmaacidophilum, and Methanobacterium espanolae. In this inventionarchaeosomes are vesicles that are prepared with lipids that include inits composition ether lipids extracted from one or more members of theArchaea, or a lipid(s) that mimics ether lipid structure(s) found inmembers of Archaea, or from one or more ether lipid(s) purified in abiologically pure form from Archaea. It will also be appreciated thatlipids (such as those made by chemical synthesis) that mimic those foundin the Archaea could also be used to make archaeosomes for the purposesstated in the current invention.

The inventors have discovered that archaeosomes are taken up byphagocytic cells to a greater extent than are conventional liposomes.Another aspect of the invention shows the improved uptake by phagocyticcells of both conventional liposomes and of archaeosomes, through theincorporation of coenzyme Q₁₀ in the respective vesicles. Incorporationof CoQ₁₀ into conventional liposomes, and archaeosomes, also allows forthe improved targeting of vesicles to specific tissues in the animal,for vesicles administered via different routes. CoQ₁₀-containingarchaeosomes with polyethylene glycol also incorporated, areparticularly effective in the targeting of orally administered vesiclesfor delivery to tissues of the spleen and liver. This would beespecially applicable for oral delivery of vaccines. Further,archaeosomes in general, are shown to be superior, compared toconventional liposomes, as carriers of antigens, resulting in improvedimmune response to the antigen administered to an animal such as ahuman. Further, it is shown that archaeosomes act as superior adjuvants,compared to conventional liposomes, resulting in an increased immuneresponse to an antigen administered to an animal such as a human. Also,the duration of the immune response, as measured by the antibody titre,can be prolonged by preparation of archaeosomes with lipids containing ahigh proportion of tetraether bipolar lipids. Finally, coenzyme Q₁₀,when used in the preparation of vesicles, is shown to improve the immuneresponse to an antigen co-entrapped either in conventional liposomesand/or in archaeosomes.

This invention will be better understood from the data given under theheading “RESULTS AND DISCUSSION”. The data therein are for illustrativepurposes only and do not limit the scope of the claimed invention.

Definitions of the Various Terms used in this Description

Antigen, an immunogen to which an animal such as a human mounts animmune response; conventional phospholipid, a glycerolipid in which thehydrocarbon chains are linked to the glycerol backbone via ester bonds;ether lipid, a glycerolipid in which the hydrocarbon chains are linkedto the glycerol backbone via ether bonds; archaeal or archaeobacteriallipid(s), lipid(s) derived from a member(s) of the class Archaea(synonymous to Archaeobacteria); liposome, closed vesicle made of lipidbilayer membranes which entrap an aqueous volume, the liposome may beunilamellar (one bilayer) or multilamellar (multiple bilayers, eachseparated from the adjoining one by aqueous spaces); conventionalliposome, a liposome made with conventional phospholipids and in somecases including a sterol and may include other compounds that areentrapped within the vesicle or associated with the bilayer membrane;archaeosome, a lipid vesicle made with one or more of the ether lipidsthat are unique to the species in the class Archaea, including thosevesicles made from any combination of lipids that includearchaeobacterial ether lipid(s) in their composition, the vesicle layerof archaeosomes may be entirely in the form of a bilayer (if madeexclusively with monopolar diether lipids or with lipid mixturescontaining diether and other monopolar lipids), or a monolayer (if madeexclusively with bipolar tetraether lipids), or a combination of monoand bilayers (if made with diether or other monpolar lipids andtetraether lipids); vesicle, liposome or archaeosome; bare antigen,antigen without adjuvant or vesicle; bare liposome/archaeosome, liposomeor archaeosome without an associated antigen; adjuvant, a substance ormaterial which when administered with an immunogen increases the immunereaction to that immunogen. The name of the archaeobacterium associatedwith the word archaeosome (e.g., M. espanolae archaeosome, orarchaeosome of from M. espanolae) indicates that the archaeosome is madewith lipids extracted from that specific archaeobacterium , and unlessstated to the contrary, from the total polar lipids (TPL) extracted fromthat archaeobacterium.

Materials and Methods

Materials

Archaeobacterial cultures were Methanospirillum hungatei GP1 (DSM 1101), Methanococcus jannaschii JAL-1 (DSM 2661), Methanococcus voltae PS(DSM 1537), Methanosarcina mazei S-6 (DSM 2053), Methanobrevibactersmithii AL1 (DSM 2375), Methanosphaera stadtmanae MCB-3 (DSM 3091),Methanobacterium espanolae GP9 (DSM 5982), Halobacterium cutirubrum (DSM669), and Thermoplasma acidophilum 122-1B3 (ATCC 27658). Cultures werecultivated according to Sprott et al. (20).

L-α-dipalmitoylphosphatidylcholine (DPPC),L-α-dimyristoylphosphatidylcholine (DMPC),L-α-dimyristoylphosphatidylglycerol (DMPG),distearoylphosphatidylcholine (DSPC), dicetylphosphate (DCP, i.e.,dihexadecyl phosphate), cholesterol (CHOL) (all these were at least 99%pure), 5(6)-carboxyfluorescein (CF), Triton X-100, n-propyl gallate,peroxidase substrate 2,2′-azino-bis (3-ethylbenzthiazoline sulphonicacid), horseradish peroxidase (1380 Units/mg), fatty acid free bovineserum albumin (BSA), coenzyme Q₁₀, vitamin E, Freund's complete adjuvant(CFA), and Freund's incomplete adjuvant (IFA) were purchased from SigmaChemical Co., Mo., USA Distearoylphosphatidylethanolamine-polyethyleneglycol 5000 conjugate (DSPE-PEG) was purchased from Avanti Polar Lipids,Inc., Alabama. Cholera toxin B subunit, from Vibrio cholerae, waspurchased from Calbiochem (La Jolla, Calif.). Cholesteryl [1-³H]hexadecyl ether (³H-chol), 52 Ci/mmole was purchased from AmershamCanada Ltd., Oakville, Ont. Silica gel G was from Macherey Nagel andCo., Duren, Germany. The LiposoFast system and 400 nm filters were fromAvestin, Inc., Ottawa, Canada. n-octyl-β-glucopyranoside (OGP) was aproduct from Calbiochem. Fetal bovine serum (FBS), and all mediacomponents including DMEM and RPM1 media, were purchased from Gibco LifeTechnologies, Inc., Grand Island, N.Y. Radioactivity counting supplieswere obtained from ICN Biomedicals Inc., Irvine, Calif. 0-chainpolysaccharide prepared from Escherichia coli 0:157:H7 was a gift fromDr. M. B. Perry (16). Immunological reagents for antibody isotyping werefrom Isotec, distributed by CedarLane Laboratories (Homby, Ont.). Serumseparator tubes were from Becton Dickinson (Rutherford, N.J.), andhorseradish peroxidase (HRP)-conjugated goat anti-mouse IgG+IgM fromCaltag (South San Francisco, Calif.).

Bacterial Lipids

Total lipids were extracted from frozen cell pastes of different speciesof archaeobacteria, and the total polar lipids (TPL) were collected asthe acetone insoluble fraction as described (20). When required, lipidsin biologically pure form were obtained from the mixture of extractedlipids by using preparative thin layer chromatography, and purity wasconfirmed by negative-ion fast atom bombardment mass spectrometry.PGP-Me(2,3-diphytanyl-sn-glycerol-1-phospho-3′-sn-glycerol-1′-methylphosphate)was purified from Halobacterium cutirubrum (9); D_(oH)PI (hydroxydietheranalog of phosphatidylinositol) and D_(OH)PG (hydroxydiether analog ofphosphatidylglycerol) were purified from M. mazei (19); and aphosphatidylinositol glycotetraether lipid of m/z 1703 daltons waspurified from lipids extracted from M. smithii. It will be recognized byone skilled, that the lipids may be extracted and/or purified by methodsother than those described here, to obtain acceptable lipids for theutility described.

Liposome and Archaeosome Preparation

For liposome and archaeosome preparation, the lipids dissolved inchloroform were dried under a stream of N₂, and placed in a lyophilizerfor 1-2 hours prior to hydration. Hydration buffer consisted of 0.5 mlof 10 mM potassium phosphate buffer, pH 7.14, containing 160 mM NaCl(PBS). Unless indicated otherwise, liposomes and archaeosomes were madeby pressure extrusion of the hydrated lipids through two stacked 400 nmfilters in a LiposoFast apparatus similar to that described by MacDonaldet al. (10) to obtain predominantly unilamellar vesicles.Archaeobacterial lipids were hydrated for 1-2 hours, or sometimesovernight, at 35° C. and the resulting multilamellar vesicles wereeither used directly (when specified) or extruded at ambienttemperature. Conventional phospholipids were hydrated (1-2 h) andextruded at 50° C. for DPPC, and at 35° C. for DMPC:DMPG:cholesterol(1.8:0.2:1.5, molar ratio) or DMPC:DMPG (1.8:0.2, molar ratio) to obtainunilamellar vesicles.

Vesicles with entrapped CF or peroxidase were prepared for in vitrophagocytosis studies, by incorporating into the hydration buffer 1.5 mMCF or 100 μg peroxidase (138 units), respectively. Typically, 10 mg ofdried TPL extracted from the specified archaeobacterium, or either ofthe conventional lipids DPPC or DMPC:DMPG:CHOL at the molar ratioindicated above, were hydrated for vesicle formation by pressureextrusion, as described above. Unencapsulated CF in CF-vesiclepreparations was removed in Sephadex G-50 columns using the minicolumncentrifugation method described by New (14). In the case ofperoxidase-vesicles, unbound enzyme was removed by centrifugation(200,000×g, max, 30 min) and the vesicles washed 4-5 times with 7 mlaliquots of PBS.

The in vitro uptake of archaeosomes prepared from the TPL extracted fromM. mazei and of conventional liposomes prepared from DPPC:CHOL (5:5molar ratio), DSPC:CHOL (5:5 molar ratio) or DSPC:CHOL:DCP (4:5:1 molarratio) was also studied using cholesteryl [1-³H] hexadecyl ether(³H-chol) as the lipidic marker. ³H-chol dissolved in chloroform, wasadded (1 μCi/mg total lipid) to the respective lipid mixture beforedrying the lipids. When required, coenzyme Q₁₀ dissolved in chloroformwas added (at 5:20 weight/weight ratio of total lipids) before dryingthe lipids. The liposomes and archaeosomes were prepared by thereverse-phase evaporation (REV) method (at 55° C. ) combined with bathsonication (at room temperature), using the method as described by New(13). ³H-chol incorporation into these vesicles was at an efficiency ofat least 95%, and these vesicles had essentially the same specificactivities. Material unassociated with the vesicles was removed bycentrifugation and washing with PBS as described above.

For in vivo tissue distribution studies, the various vesicles with³H-chol as the lipidic marker, were prepared as described below. Whenindicated, these vesicles also contained coenzyme Q₁₀ and/ordistearoylphosphatidylethanolamine-polyethylene glycol 5000 conjugate(DSPE-PEG), which were added to the rest of the lipids before drying.Archaeosomes (M. mazei TPL), CoQ₁₀-archaeosomes (M. mazei TPL:CoQ₁₀ at amolar ratio of 8:2 using the average molecular weight of the TPL as1000), PEG-archaeosomes (M. mazei TPL plus DSPE-PEG at 7% molar ratio ofthe total lipids), and PEG-CoQ₁₀-archaeosomes (M.mazei TPL:CoQ₁₀ at amolar ratio of 8:2 plus DSPE-PEG at 7% molar ratio of the total lipids)were prepared with ³H-chol (specific activity of 52 μCi/mmole) added at10 μCi per mg of total lipid used for vesicle preparation. Vesicles wereprepared by the REV-bath sonication method described above for preparing³H-chol-labeled vesicles for in vitro studies.

Conventional liposomes (DSPC:DCP:CHOL at a molar ratio of 4:1:5),CoQ₁₀-conventional liposomes (DSPC:DCP:CHOL:CoQ₁₀ at a molar ratio of3:1:4:2), PEG-conventional liposomes (DSPC:DCP:CHOL at a molar ratio of4:1:5 plus DSPE-PEG at 7% molar ratio of the total lipids), andPEG-CoQ₁₀-conventional liposomes (DSPC:DCP:CHOL:CoQ₁₀ at a molar ratioof 3:1:4:2 plus DSPE-PEG at 7% molar ratio of the total lipids) wereprepared with ³H-chol as described above for archaeosomes.

For in vivo studies with archaeosomes (prepared from the TPL, or a lipidobtained in a biologically pure form from the indicatedarchaeobacterium) and conventional liposomes (prepared from DMPC:DMPG at1.8:0.2 molar ratio, or DMPC:DMPG:CHOL at 1.8:0.2:1.5 molar ratio, orDMPC:DCP:CHOL at 7:1:2 molar ratio) containing entrapped antigen, thevesicles were prepared by pressure extrusion (at room temperature forarchaeobacterial and at 35° C. for DMPC-containing lipids, using 400 nmpore size filters). About 20 mg dry weight of the dried lipid(s) washydrated in 1 ml PBS containing either BSA (5 mg/ml), Cholera B toxinsubunit (0.5 mg/ml), or O-chain polysaccharide (18 mg/ml). To entrap BSAin DSPC:DCP:CHOL (4:1:5, molar ratio), in DSPC:DCP:CHOL:CoQ₁₀ (3:1:4:2,molar ratio), and M. mazei TPL:CoQ₁₀ (8:2, molar ratio), the vesicleswere prepared by the REV (at 55° C.)-sonication method described above.The BSA was encapsulated in these vesicles by the DRV method asdescribed by New (13). The antigen that was not entrapped/associatedwith the vesicles was removed by centrifugation and washing as describedfor peroxidase-vesicles.

Vitamin E was incorporated into archaeosomes by dissolving in CHCl₃, 1mg vitamin E and 20 mg of T. acidophilum TPL. The CHCl₃ was removed, thedried lipids and vitamin E hydrated with PBS, and vitamin E-archaeosomesprepared by pressure extrusion, as described above.

It will be understood by one skilled in the art that the preparation ofliposomes and archaeosomes of this invention and theassociation/incorporation of other compounds in the respective vesiclesis not limited to the illustrated methods, and may also be made usingother methods of record in the published literature. The vesicles ofthis invention may also be prepared and used in conjunction withadditional compounds known to have beneficial properties, such asserving as an additional adjuvant.

Vesicle Characterization

The mean vesicle diameters were determined by number-weighted sizedistribution using a Nicomp particle sizer, model 370 (Nicomp, SantaBarbara, Calif.).

Peroxidase activity in 5 to 35 μg dry weight of liposomes orarchaeosomes was assayed in 1 ml reaction mixtures containing 45 mMcitric acid, pH 4.0, 2.2 mM H₂O₂, 0.2 mM 2,2′azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), in the presence of 0.5% (w/v)of the detergent OGP. Reaction rates were recorded at 23° C. with aColeman 575 recording spectrophotometer.

CF was quantitated with a spectrofluorometer set at an excitationwavelength of 470 nm and an emission wavelength of 520 nm.

The amount of protein incorporated into or associated with archaeosomesand liposomes to be used for immunizations was quantitated by SDSpolyacrylamide gel electrophoresis (PAGE), wherein the lipids separatefrom the protein antigen. Densitometry of stained gels was done with agel documentation system from Canberra Packard Canada, and quantitatedfrom standard curves prepared by loading 0.025 to 2 μg of the protein ofinterest onto each lane. The amount of protein incorporated/associatedwith the vesicles was compared on the basis of salt free dry weights ofthe liposomes or archaeosomes, respectively.

To determine the amount of coenzyme Q₁₀ entrapped in the lipid layer ofvesicles, the vesicles were dissolved in chloroform and the absorbencywas measured at 278 nm. A standard curve was prepared using purified Q₁₀dissolved in chloroform, and a concentration of 10 μg CoQ₁₀/ml gave anabsorbency of 0.2.

Murine Peritoneal Macrophages and Cell Lines

Murine peritoneal macrophages, harvested from 12-16 week old femaleBalb/c mice as previously described (23), were maintained in RPMI-1640medium supplemented with 25 mM HEPES buffer, 2 mM L-glutamine, 40 μg/mlgentamicin, and 10% FBS. HEp-2 (human laryngeal epithelial carcinomacell line, ATCC #CCL 23), and HeLa (human cervical epitheliod carcinomacell line, ATTC #CCL 2), and the monocyte-macrophage cell line J774A.1(ATCC #TIB 67) were obtained from the American Type Culture Collection,Rockville, Md. These three cell lines were maintained in the same mediumas were the peritoneal macrophages. EJ/128 cells (human uroepithelialcarcinoma cell line) were obtained from Dr. Derek Duke, Imperial CancerResearch Fund, London, England. EJ/128 cells were maintained in DMEMsupplemented with 2 mM L-glutamine, 40 μg/ml gentamycin and 10% FBS. Allcells were sustained at 37° C. in a humidified atmosphere containing 5%CO₂ in air.

Peroxidase Assay for in vitro Vesicle Binding to Cells

Archaeosomes or liposomes containing encapsulated peroxidase wereincubated with different cell types to quantitate the respective vesiclebinding to mammalian cells. Peritoneal macrophages were prepared asdescribed previously to give confluent monolayers (23). The cell lineswere seeded at 50% of confluence onto tissue culture plates (Nunc Inc.,Naperville, Ill.). All the cell lines, in their respective media, wereplated onto 96-well tissue culture plates to form confluent monolayers.The next day, the cells were washed twice with the respective growthmedium to remove nonadherent cells and then incubated (50 min at 37° C.)in fresh medium supplemented with the various vesicle preparations. Theadherent cells were then washed 5× with 200 μl ice-cold PBS containing0.9 mM CaCl₂, to remove unbound vesicles. The vesicles and cells weresubsequently lysed using 100 μl of 0.5% OGP, and 100 μl of 2×concentrated substrate solution was added. After 30 min incubation at25° C., the colour reaction was measured at 405 nm using a DynatechMR5000 microplate reader (Chantillym, Va.). The readings were convertedto μg of vesicles bound per mg of cell protein, based on the amount ofperoxidase that was encapsulated in the respective vesicles used in thebinding assay.

Fluorescence Microscopy Assessment of in vitro Vesicle Binding to Cells

Fluorescence microscopy was performed to visualize archaeosome orliposome binding to several cell types. For these studies, CF wasentrapped within the vesicles at a concentration of only 1.5 mM, toavoid self-quenching. Peritoneal macrophages and the various cell lineswere seeded at approximately 20% of confluence onto 24-well tissueculture plates (Nunc Inc., Naperville, Ill.). The next day the cellswere washed twice with ice-cold PBS containing 0.9 mM CaCl₂ to removenon-adherent cells and incubated with the various CF-containing vesiclepreparations (40 μg/ml) for 30 min at 37° C. The cells were washedextensively with ice-cold PBS and then preserved in PBS containing 1%formaldehyde and 0.1% n-propylgallate (fluorescence fading inhibitor).The adherent cells and bound CF-vesicles were observed using an OlympusIMT-2 inverted microscope fitted with an IMT2-RFL reflected lightfluorescence attachment and a 35 mm photomicrographic camera.

³H-chol Assay for in vitro Vesicle Uptake

These uptake studies were performed essentially as described above, butused liposomes and archaeosomes prepared to contain tritiatedcholesteryl [1-³H] ether (³H-chol) as a tracer. One micro curie (1μCi=37 kBq) of ³H-chol was added per milligram of lipid before makingthe vesicles. Radioactive label taken up by J774A.1 macrophages wasassayed and calculated on the basis of the amount of macrophage protein,according to Makabi-Panzu et al. (11).

Inhibition of Macrophage Functions

Adherent macrophages were treated to inhibit their phagocytic functions,and the influence of this on archaeosome binding/uptake was monitored.J774A.1 macrophages were seeded in 24-well tissue culture plates to formsemi-confluent monolayers. The next day the cells were washed twice withice-cold PBS. CF-containing archaeosomes prepared from the TPL of M.hungatei were added to the macrophages at a concentration of 0.04 mg/mland the samples were allowed to incubate for 30 min at 37° C. The cellswere then washed extensively with ice-cold PBS containing 0.9 mM CaCl₂to remove non-adherent archaeosomes and the cells were resuspended infresh media alone, in media containing inhibitors, or in media cooled to4° C. The cultures were then incubated for up to 300 min at 37° C. (or4° C., as indicated). At required time intervals, macrophages wereexamined by fluorescence microscopy to obtain an estimate of the extentof CF release from the archaeosomes.

For assays using formaldehyde-fixed macrophages, the cells were firstincubated with archaeosomes and washed as described above. Then, 0.5 mlof a 0.5% formaldehyde solution was added to each well and the cellswere incubated at room temperature for 15 min. The fixative was removedby extensive washing with PBS and media, prior to the start ofincubation for up to 240 min.

To examine the effects of inhibition of the polymerization ofcytoskeletal components of the cells on archaeosome uptake, macrophageswere incubated in media containing 10 μg/ml each, of cytochalasins B andD (in a final concentration of 1% dimethyl sulfoxide) beginning 30 minprior to the addition of archaeosomes, and continuing to the end of thetime trial period. Macrophages were also incubated under the sameconditions in media containing 1% dimethyl sulfoxide (DMSO) to evaluateany possible effect of DMSO.

In vivo Tissue Distribution of Vesicles

Inbred female Balb/c mice were purchased from Charles River Laboratories(St. Constant, Quebec) and maintained in the Animal Care Unit at theNational Research Council of Canada.

Various types of vesicles containing ³H-chol were administered to Balb/cfemale mice (1 mg total lipids per mouse, ca. 50 mg/Kg of body weight),in triplicate, via the p.o. route using -intra gastric intubation todeliver directly to the stomach, via the i.m. route, via the s.c. route,or via the i.v. route (injected in the tail vein). The mice were 6-8weeks old at the time of vesicle administration. The mice were allowednormal access to food and water. After 24 or 48 hourspost-administration, the mice were euthanised, plasma and organs werecollected, and the radiotracer counts associated with the organ tissueswere determined in an LKB 1217 Rackbeta liquid scintillation counter(Pharmacia Canada, Baie d'Urfé, Que.) using methods described previously(12). The distribution of the radiotracer per gram of the various organtissues (or per ml plasma) was calculated and expressed as a percentageof the total radiotracer count administered to the animal at time zero(% dose/g tissue).

Immunization Protocols

The immunization strategies were similar for each of the differentexperiments. For each experiment, details of the amounts and types ofantigen/adjuvant injected, and the time intervals between injections isdescribed in the appropriate figure legends. Freund's adjuvant (FA)encompasses Freund's complete adjuvant (CFA) and Freund's incompleteadjuvant (IFA) used at 62.5% strength in PBS. For each antigen/adjuvantpreparation three mice were injected (unless indicated otherwise) i.p.,i.m., or s.c. with antigens encapsulated in archaeosomes or liposomes(diluted in sterile PBS, pH 7.1), antigens emulsified in CFA, or withantigens diluted in PBS alone (final volume, 0.2 ml/mouse). For thesecond immunization, mice were injected with antigens encapsulated inarchaeosomes or liposomes, or emulsified in IFA, or diluted in PBS. Forexperiments where a third or fourth injection was required, antigenswere injected either encapsulated in the respective vesicle type or asbare antigen diluted in PBS. Mice were 6-8 weeks of age at the time ofthe first immunization.

Mice were bled from the tail veins usually four days after eachinjection. The blood was allowed to dot, and cells removed from theserum by centrifuging in serum separator tubes.

In vivo Administration

Administration of the vesicles which carry the antigen, drug or otheringredients is by the customary routes, and may be used with additionalsubstances, such as carbonate buffer when given orally. The requireddose of antigen will vary depending on the antigen used and on the routeof administration, but is about 1 to 50 μg per dose. Entrapments ofprotein antigens in the vesicles range from about 0.9-208 μg/mg dryweight of vesicles, or 0-chain polysaccharide up to 500 μg/mg vesicles.Coenzyme Q₁₀ may be incorporated from 0 up to about 0.23 mg/mg vesicles,and vitamin E from 0 up to about 0.2 mg/mg vesicles with 0.05 mg/mgpreferred. Generally, the dosage of vesicles is in the range of 4 to 73mg/kg body weight, based on a weight of 25 g/mouse.

Nevertheless, it may be necessary, under certain circumstances, todeviate from the amounts mentioned, and in particular to do so as afunction of the body weight or of the nature of the administrationmethod, the nature of its formulation, the animal species, and the timeor interval over which the administration takes place. Thus, it can insome cases be sufficient to manage with less than the above-mentionedamount, whereas in other cases more than the upper limit mentioned maybe required.

Enzyme-linked Immunosorbent Assays

The humoral response was measured by the indirect enzyme-linkedimmunosorbent assay (ELISA) using a variety of solid-phase adsorbedantigens. BSA, Cholera B toxin subunit, or E. coli 0:157:H7lipopolysaccharide was diluted in distilled water (15 μg/ml finalconcentration) and 100 μl/well was dried (overnight incubation at 37°C.) to coat the ELISA microplate wells. All indirect ELISA assays wereperformed using standard methods. Dilutions of antibody-containing serawere used as the first antibody and a 1/1500 dilution of HRP-conjugatedgoat anti-mouse IgG+IgM as the detection antibody.

The isotypes of anti-BSA antibodies raised in mouse sera were assayed byan ELISA method. Wells were coated with BSA (as above). Dilutions ofeach serum were titred with peroxidase-coupled sheep anti-mouse IgG1,IgG2a, IgG2b, IgG3, and IgM. All ELISA data are reported as the means ±sample standard deviations.

Results and Discussion

Construction and Characterization of Archaeosomes and Liposomes

Peroxidase encapsulated within vesicles was used to quantitate therelative uptake of the various types of archaeosomes and of conventionalliposomes, by eukaryotic cells. The amounts of each vesicle type takenup by adherent cells seeded onto culture wells could be quantitated bymeasuring peroxidase activity following lysis/permeabilization of bothcells and associated vesicles by detergent.

Archaeosomes were prepared from the TPL extracted from severalarchaeobacteria, and conventional liposomes prepared from twoconventional phospholipid formulations that have been frequently used inthe prior art (2, 17). The percentage of the total peroxidase which wasexposed on the vesicle surface could be estimated by comparing the rateof the enzyme reaction in whole vesicles and in OGP-permeabilizedvesicles (Table 1).

Except for the data in FIG. 2, we prepared intermediate-sized vesiclesof approximately 200 nm diameter by pressure extrusion of hydratedmultilamellar liposomes through filters of 400 nm pore size. Thisresulted in populations of vesicles with the size range distributionsdefined in Table 1. The distributions were quite narrow except for DPPCliposomes, which consisted of two differently sized populations.

Binding of Archaeosomes and Conventional Liposomes to Cells

Binding and uptake of the immunogen by macrophages or other antigenprocessing/presenting cells is the basis for the induction of an immuneresponse. Peroxidase and fluorescence assays were used to quantitate andcompare the binding of several types of archaeosomes and of conventionalliposomes to two phagocytic and three non phagocytic cell lines. Theresults are illustrated in FIG. 1, and in Tables 2 and 3. The uptake ofarchaeosomes by the two macrophage lines (murine peritoneal and J774A.1macrophages) was several times greater than the conventional liposomes.Compared with macrophage cell lines, the uptake of archaeosomes and ofconventional liposomes by non-phagocytic cell lines (Hep-2, HeLa, EJ/28)was substantially lower.

Large, multilamellar vesicles (0.5 to 3 μm) prepared from the polarlipids of M. mazei or from DMPC:DMPG:cholesterol gave trends similar tothose obtained with the smaller vesicles, i.e. greater uptake ofarchaeosomes than conventional liposomes by murine peritonealmacrophages, and by J774A.1 cells; lesser uptake of both vesicle typesby the non- phagocytic HEp-2 cell line (FIG. 2).

These data, using encapsulated peroxidase or fluorescent dye as markers,illustrate the enhanced phagocytosis of archaeosomes compared withconventional liposomes. The potential advantages for using archaeosomesfor delivery of compounds to macrophages is discussed elsewhere in thissubmission.

Effect of Inhibition of Macrophage Functions on Archaeosome Binding

To determine if archaeosome populations were actively phagocytosed, asopposed to adhered to the surface, macrophages were treated withinhibitors of phagocytosis (Table 4). As demonstrated by the controlpopulation of untreated macrophages exposed to archaeosomes, the amountof fluorescence associated with these macrophages was seen to decreasemarkedly over time. This is consistent with internalization of thearchaeosomes, followed by release of the encapsulated dye due todegradation of the vesicle. In contrast, treatments which are typicallyknown to inhibit membrane flow and thus phagocytosis, such as decreasedtemperature, the addition of cytochalasins, or fixation with formalin,resulted in little or no decline in the fluorescence (present due toinitial binding of vesicles, before inhibition of phagocytosis) of themacrophages over the time periods studied. This indicates that theinhibitors prevented internalization of the liposomes into themacrophages. In addition, the time-dependent decline in fluorescenceupon readjustment of the temperature of the culture medium from 4° C.back to 37° C. demonstrated that the macrophages recovered theirphagocytic abilities. An appropriate model encompasses an initialbinding to the cell surface, followed by phagocytosis, and degradationof the internalized vesicles.

Use of Coenzyme Q₁₀ to Enhance the Uptake of Vesicles by Cell Lines

Coenzyme Q₁₀ was entrapped into archaeosomes prepared from the TPL of M.mazei, which are known to be anionic (19), and into anionic conventionalliposomes (DSPC:CHOL:DCP), with relatively high entrapment efficiencies(Table 5). However, compared with anionic lipid mixtures, even higherentrapment efficiencies were obtained with vesicles prepared withneutral lipid mixtures DPPC:CHOL and DSPC:CHOL. The loading ratios shownare representative of those used in subsequent experiments (Tables 6-7).

The uptake by macrophages, of archaeosomes and of conventional liposomeslacking coenzyme Q₁₀ is shown as a function of time using ³H-chol as thetracer marker (Table 6A). At 37° C., at the indicated times, it can beseen that M. mazei archaeosomes are taken up substantially better thanall formulations of conventional liposomes, as was also shown in Table2. The data in Table 6A serve as control values to assess the effect onvesicle uptake of incorporating coenzyme Q₁₀ into the vesicles (Table6B). These data show that the cellular accumulation of all vesicletypes, by the macrophages, was markedly enhanced when the vesiclescontained coenzyme Q₁₀. This enhancing effect of coenzyme Q₁₀ wasseveral times higher with the archaeosomes than with the conventionalliposomes. Comparable profiles of accumulation and the enhancing effectsof coenzyme Q₁₀ entrapment on the uptake of archaeosomes and ofconventional liposomes, was observed at all lipid concentrations tested(compare Tables 7A and 7B).

These results clearly indicate the potential for inclusion of coenzymeQ₁₀ into the vesicles for increased targeting of archaeosomes and ofconventional liposomes to various cell types. An increased uptake ofvesicles containing coenzyme Q₁₀ by macrophages (antigen processingcells, and the sites for some infectious agents) dearly indicatesapplication in delivery of antigens, and drugs (including antiviral andantimicrobial agents). Another application is to deliver thewater-insoluble drug (coenzyme Q₁₀) to mammalian cells, via liposomesand archaeosomes.

Archaeosomes prepared from vitamin E and the TPL of T. acidophilum, asan example, could be made. Incorporation of 1 mg vitamin E into 20 mglipids resulted in archaeosomes with the expected size range of 220±88nm following extrusion through 400 nm pore size membranes.

In vivo Tissue Distribution of Vesicles

The distribution, into various tissues, of the ³H-chol lipidicradiotracer marker incorporated into the vesicle layer was used topredict the delivery of the vesicles and/or the associated/encapsulatedcompounds to the specific organ tissues. Cholesteryl [1-³H] ether is anon-metabolized radiotracer (marker) suitable for studying tissuedistribution of lipidic vesicles in animals such as mammals (15).

The tissue distribution profiles of label from conventional liposomesand CoQ₁₀-conventional liposomes administered by various routes showedthat these were generally similar (FIGS. 3A and 3B). However, the majordifference was that in orally administered CoQ₁₀-conventional liposomes,no accumulation of the label (at 24 h) was seen in any of the tissuesexamined, except for in the spleen.

PEG conjugated to lipids, such as DSPE, has been used in conjunctionwith conventional lipids to prepare vesicles with altered surfaceproperties, to evade capture by the cells of the reticuloendothelialsystem (predominantly present in the spleen and liver), and henceprolong the circulation half life in the blood. Such liposomes have beencalled sterically stabilized liposomes (24). The tissue distributionprofile of PEG-conventional liposomes (FIG. 4A) was somewhat similar tothat of conventional liposomes (FIG. 3A). However, A was surprising tosee that when CoQ₁₀ was incorporated into the PEG-conventional liposomeswhich were administered orally, there was an increased accumulation ofthe marker (24 hours post administration) into spleen, liver, intestine,kidney and lung tissues (FIG. 48). The combined accumulation in thespleen and liver was about 8-fold greater. With the i.v. route ofadministration, the PEG-CoQ₁₀-conventional liposomes showed no presenceof the marker in the spleen (FIG. 4B).

Except for the accumulation in the intestine and kidneys, the tissuedistribution profiles of M. mazei archaeosomes (FIG. 5A) showeddifferences from those with conventional liposomes (FIG. 3A). When M.mazei archaeosomes were administered via i.m., s.c., or i.v. routes, thetissue distribution profile (at 24 h), with and without CoQ₁₀ entrappedin the vesicles, was similar except for that in the intestines (FIGS. 5Aand 5B).

It was very surprising to see that in the case of orally administeredPEG-CoQ₁₀-archaeosomes, the accumulation of the label (24 hpost-administration) in the spleen was up to 80-fold higher than thatseen with PEG-archaeosomes (FIGS. 6A and 6B). This accumulation in thespleen represented about 40% of the total administered label. Thecombined level of incorporation of orally deliveredPEG-CoQ₁₀-archaeosomes in the liver and spleen, was about 5-fold betterthan the highest level obtained (FIG. 4B) with any combination usingconventional liposomes. For i.m. and i.v. administeredPEG-CoQ₁₀-archaeosomes, the accumulation of label in the spleen wassignificantly lower than that obtained in the absence of CoQ₁₀ (i.e., inPEG-archaeosomes).

The 48 hour tissue distribution profile of label from orallyadministered PEG-CoQ₁₀ conventional liposomes was similar to thatobserved at 24 hours (FIG. 4B). However, the 48 hour tissue distributionprofile of label from orally administered CoQ₀-archaeosomes, whether ornot also associated with PEG, indicated that the amount in spleen, liverand intestines had declined to about 0.001%, each, of the administereddose, from the respective higher levels (FIGS. 5B and 6B) at 24 hours.At 24 hour post-administration there was no detectable label in theserum, irrespective of the vesicle type or the route of vesicleadministration (FIGS. 3-6).

The data in these figures show that a significant enhancement in theefficacy of the delivery/accumulation of orally administeredconventional liposomes, to the spleen and liver (the major sites for theantigen processing cells of the immune system), can be achieved byincorporation of CoQ₁₀ in these vesicles. The delivery to the spleen andliver by orally administered vesicles can be further enhanceddramatically, by using PEG-CoQ₁₀-archaeosomes. It is also evident thatby using different combinations of vesicle types, with and withoutentrapped CoQ₁₀, one can obtain various different tissue distributionprofiles. This offers a means to alter the tissue targeting fordifferent applications in the fields of medicine.

Immune Responses

The enhanced uptake of archaeosomes by phagocytic cells, compared tothat of conventional liposomes, suggested that archaeosomes may besuperior as adjuvants and/or carriers of antigens for raising an immuneresponse to an immunogen. This was found to be the case in animal modelstudies using mice.

Compared to control mice receiving the bare antigen, the antibody titerin sera from mice immunized with cholera toxin B subunit was found to besignificantly higher when the antigen was entrapped in archaeosomes ofM. smithii, and this response was even comparable to that observed withFreund's adjuvant (FIG. 7).

A comparison of the adjuvant/antigen carrier properties of archaeosomesand of conventional liposomes was made using BSA as the antigen (FIG.8). The immune response to BSA was markedly enhanced when it wasencapsulated in archaeosomes, and the results were again comparable, insome cases, to that achieved with Freund's adjuvant. In contrast, allthree conventional liposome types yielded substantially lowerimmunostimulatory effects.

The number of boosts needed to achieve a circulating antibody titrecomparable to that achieved with Freund's adjuvant, was determined usingthe BSA antigen entrapped in archaeosomes of M. smithii (FIG. 9). Whenarchaeosomes (with the antigen encapsulated) were given only once,followed by a boost with bare antigen, a considerable immune response,comparable to that with Freund's adjuvant, was achieved.

It was noted that the stimulation of the immune response with certainarchaeosomes (for example M. espanolae) administered i.p. required thatthe BSA antigen be entrapped in the vesicle, whereas with otherarchaeosomes (for example T. acidophilum) entrapment was not aprerequisite (Table 8). In another example using Cholera toxin B, theimmunostimulatory effect was greater when the antigen was entrapped inthe archaeosome (FIG. 7). However, when the BSA antigen was administeredvia the i.m. route, association with the archaeosome was required for agood immune response (Table 9). Hence, whether or not the antigen neededto be entrapped or associated with the vesicle would depend on the routeof administration and the lipid composition of the vesicles.

For use in humans, other mammals, and other life-forms such as avian andmarine species, it is important to obtain the immune reaction followingimmunization via normally acceptable routes of delivery. Comparison ofs.c., i.m., and i.p. immunizations established that the humoral responsecould be elevated by archaeosomes administered via various routes (Table10).

The small adjuvant effect observed with DMPC:DMPG conventional liposomescan be improved by the inclusion of increasing amounts ofarchaeobacterial lipids in the lipids used for vesicle formation. Thisis illustrated in Table 11 using vesicles made with DMPC:DMPG:M. smithiiTPL, where the amount of TPL was varied from 0 to 100% in the mixtureused to form vesicles and encapsulate the BSA antigen. An enhancement ofthe humoral immune response to the protein was seen by the inclusioninto the vesicles of as little as 10% of archaeobacterial lipids, andprogressive improvements seen up to 100%.

It is further shown that archaeosomes may be prepared from lipidsobtained in a biologically pure form from archaeobacteria, and thatthese archaeosomes also have the ability to enhance the immune responseto an antigen (Table 12). While all archaeosomes prepared from purelipids produced a positive adjuvant effect, PGP-Me was superior. Thismay be explained by the novelty of the doubly charged anionic head groupof PGP-Me.

The duration of the immune response to the encapsulated antigen ismaintained longer when tetraether lipids are used, compared to dietherlipids, to prepare the archaeosomes (Table 12). The decline in theantibody titre between 35 and 55. days was 40 to 46% in the case ofFreunds adjuvant and the 3 diether lipid archaeosomes, whereas thedecline was only 25% for the tetraether archaeosome.

Comparison of M. smithii archaeosomes prepared from the total lipidextract (includes neutral lipids) with those prepared from the totalpolar lipid extract (neutral lipids removed), established that theimmune response to BSA. (anti-BSA antibody titres) administered i.p. was63 and 91% respectively, of that found with Freund's adjuvant The amountof antigen administered, the immunization protocols and antibody titreswere conducted as detailed in Table 11.

Archaeosomes with encapsulated/associated BSA elicited antibodies ofvarious isotypes (FIG. 10) when administered to mice. The presence ofIgG isotypes of the 2a/2b class are important indicators of protectiveimmunity and of a cell-mediated immune reaction. Hence, archaeosomeselicit not only a humoral response to the antigen being carried, butalso a cell-mediated response is expected.

Mice were immunized (days 0 and 14), i.p., with conventional liposomes(DSPC:DCP:CHOL) containing 15 μg BSA per dose, with and without Q₁₀incorporated (see Table 5 and 6B for compositions). Sera collected onday 18 when titred for antibodies (IgG+IgM) had a significantly higheranti-BSA antibody titre (at the 95% confidence limit) when the liposomescontained CoQ₁₀.

Carbohydrates are known to be poor immunogens even when administeredwith known adjuvants. In addition to enhancing the immune response toprotein antigens, it is shown here that the O-chain polysaccharideantigen from E. coli 0:157:H7 is immunogenic when associated with M.smithii archaeosomes. For these studies, Balb/c mice received 50μg/injection (i.p.) of bare O-chain polysaccharide or 50 μg/injection ofthe same antigen encapsulated into 0.1 mg of M. smithii archaeosomes(TPL). One injection was given on day zero and blood collected on day 18for titration of IgG+IgM antibodies. The bare antigen produced anA_(410 nm) reading of 0.004 compared to 0.231 with archaeosomes,representing an increase of about 58-times.

The dramatic increase in the immune response to antigens associated witharchaeosomes, observed in this study, was unexpected and contrary to theexpectations suggested from prior art disclosures using ether lipids.Indeed, Shek et al. (17) showed that vesicles made with dialkyl-etherphosphatidylcholines, with BSA as the entrapped antigen, were lessefficient in eliciting an immune response in mice compared to liposomesmade with the diacyl-ester phosphatidylcholine. In addition to enhancedadjuvant effects, few boosts are required with archaeosomes. Moreover,the storage stability (shelf life) of archaeosomes is long (20).

In vitro cytotoxicity studies with several phagocytic and non phagocyticcell lines described earlier indicated that neither archaeosomes norconventional liposomes had any significant adverse effects, as assessedfrom cell viability assays, even when the vesicles were tested at lipidconcentrations well above saturation. In mouse models, there were nosigns of gross toxicities such as granulomas at sites of injection,gross changes in the key body organs or deaths. Anti-archaeosome lipidantibodies are not detected in the mouse sera obtained in FIG. 8.Further, there are indications that molecules with ether bonds can beslowly metabolised and eliminated from the body (21). The in vitrophagocytosis data presented in our disclosure also indicate thatarchaeosomes are at least sufficiently destabilized/degraded in themacrophages to release entrapped protein and fluorescent dye markers.The in vivo data on immune response also support that the archaeosomescontaining encapsulated antigens are destabilized/degraded to allowpresentation of the antigen to the antigen processing cells of theimmune system.

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TABLE 1 Characteristics of peroxidase-archaeosomes andperoxidase-liposomes used in binding assays.¹ Mean Diameter Lipid sourcefor (nm ± standard Peroxidase Activity² vesicles deviation) No DetergentOGP M. mazei 189 ± 63 7.7 32.3 M. hungatei 218 ± 73 4.9 39.2 M.jannaschii 192 ± 69 3.5 34.7 M. stadtmanae 170 ± 63 10.3 31.6 M. smithii200 ± 77 4.3 32.9 M. voltae 206 ± 65 3.3 26.3 T. acidophilum 184 ± 854.1 70.4 DPPC    324 ± (84)³ 1.4 48.4 DMPC:DMPG:CHOL 204 ± 72 0.69 31.8¹Pressure extruded vesicles were prepared from conventional lipids orfrom the total polar lipids of Archaeobacteria: Methanósarcina mazei,Methanospirillum hungatei, Methanococcus jannaschii, Methanosphaerastadtmanae, Methanobrevibacter smithii, Methanococcus voltae, andThermoplasma acidophilum. ²Activity is expressed as change inabsorbency/min/mg dry wt vesicles. ³Consisting of two populations ofliposomes of 324 nm (84%, number weighted) and 2833 nm (16%, numberweighted).

TABLE 2 Comparative in vitro uptake (μg/mg cell protein) of archaeosomesand conventional liposomes to murine peritoneal macrophages and avariety of cell lines.¹ Murine Lipid source for Peritoneal J774A.1 EJ/28vesicles Macrophages Macrophages HEp-2 cells HeLa cells cells M. mazei36.4 ± 0.3 32.3 ± 0.6 3.5 ± 0.3 1.7 ± 0.1 1.3 ± 0.3 M. hungatei 32.9 ±1.7 21.3 ± 1.2 0.7 ± 0.0 0.2 ± 0.0 1.3 ± 0.1 M. jannaschii 34.9 ± 3.024.6 ± 0.3 0.2 ± 0.0 1.4 ± 0.1 2.2 ± 0.1 M. stadtmanae 19.8 ± 0.5 20.4 ±0.5 2.3 ± 0.3 1.4 ± 0.1 2.1 ± 0.0 M. smithii 43.2 ± 1.8 38.6 ± 1.4 1.1 ±0.1 0.3 ± 0.0 2.3 ± 0.1 M. voltae 33.8 ± 0.2 23.8 ± 0.4 2.5 ± 0.0 1.2 ±0.0 1.9 ± 0.1 T. acidophilum 13.5 ± 3.3 not done 0.7 ± 0.5 0.4 ± 0.3 1.1± 0.6 DPPC  0.8 ± 0.1  2.7 ± 0.1 0.6 ± 0.0 0.3 ± 0.0 1.1 ± 0.1DMPC:DMPG:CHOL  6.0 ± 0.3  7.2 ± 0.2 0.2 ± 0.0 0.4 ± 0.0 2.1 ± 0.0¹Peroxidase-vesicles were assayed for binding to cell lines adhered toculture wells. For comparison purposes, the extent of colour reactionwas corrected for the amount of enzyme entrapped within each vesiclepreparation. Absorbency values were corrected by subtracting a blankobtained by adding all reagents to blank wells (no cells). Values arethe average of duplicate samples ± sample standard deviation.

TABLE 3 Relative uptake of archaeosomes and conventional liposomes byvarious cell lines.¹ Vesicles Mean Diameter added per Relative Binding³Lipid source for (nm ± standard assay well J774A.1 HEp-2 HeLa EJ/28vesicles deviation) (μg) Fluorescence² cells cells cells cells M. mazei143 ± 60 120 0.153 10 1 1 1 M. espanolae 205 ± 71  88 0.136 10 1 2 2DMPC:DMPG:CHOL 223 ± 84 134 0.235  3 1 1 1 ¹Vesicles contained CF.²Emission from 10 μl vesicles diluted into 190 μl PBS, to indicate therelative fluorescence of vesicle suspensions used in the uptake assays.Fluorescence was enhanced only 5 to 12% in the presence of 0.5% OGP.³Assessed by fluorescence microscopy, where intense fluorescence fromvesicles adhered to the cells is given a value of 10, and very weakbackground fluorescence is shown as 1.

TABLE 4 The effect of inhibitors on the phagocytosis of M. hungateiarchaeosomes by J774A.1 macrophages.¹ Relative binding/degradation ofliposomes by macrophages at different intervals (min)² Treatment 0 30 60120 180 240 300 360 None 10 8 5 3 2 1 N/D N/D Cytochalasins B + D 10 1010 8 8 7 N/D N/D DMSO 10 10 5 2 2 1 N/D N/D Formaldehyde 10 10 10 10 1010 N/D N/D 4° C. 10 10 10 10 10 10 N/D N/D 4 C. → 37° C³ 10 10 10 10 7 52 1 ¹Archaeosomes with entrapped CF were prepared by pressure extrusionof the total polar lipids from M. hungatei. N/D, not determined.²Assessed by fluorescence microscopy, where intense fluorescence isgiven a value of 10 and weak background fluorescence is 1. ³Temperatureshifted to 37° C. at 120 min.

TABLE 5 Incorporation of coenzyme Q₁₀ into M. mazei archaeosomes andconventional liposomes.¹ Starting ratio Q₁₀ Loading Q₁₀:lipid EntrapmentRatio Lipid source for vesicles (mg:mg) (%) (mg Q₁₀/mg lipid) M. mazei5:20 62.1 ± 1.1 0.155 DPPC:CHOL 5:20 91.5 ± 1.1 0.230 (5:5, molar ratio)DSPC:CHOL 5:20  86.1 ± .085 0.215 (5:5, molar ratio) DSPC:CHOL:DCP 5:2053.7 ± 1.7 0.135 (4:5:1, molar ratio) ¹Vesicles were prepared byREV-bath sonication, and were of an average diameter of 100 nm. The samelipid compositions were used for Tables 6-7. The % entrapment and theloading ratio were calculated from the starting amounts of Q₁₀ andlipids. A 100 % recovery of the lipids was assumed.

TABLE 6A Uptake of M. mazei archaeosomes and conventional liposomes byJ774A.1 macrophages as a function of time, using ³H-chol as the marker.¹Lipid source for Uptake (cpm)² vesicles 15 min 30 min 45 min 60 min M.mazei 1786 ± 80  2737 ± 92  2957 ± 247  3570 ± 68  DPPC:CHOL 290 ± 48  367 ± 39.4 430 ± 70  334 ± 73 DSPC:CHOL 259 ± 9  311 ± 23 643 ± 108228 ± 25 DSPC:CHOL:DCP 570 ± 89 603 ± 25 694 ± 222 589 ± 28 ¹The valueswere determined by incubating cells with 25 μM of vesicles for theindicated incubation times. An average molecular weight of 1000 was usedto calculate the moles of lipids of M. mazei. Each culture well receiveda total volume of 5 ml; 260, 500 cpm in 125 nmoles vesicles (Tables6-7). ²cpm of accumulated lipid per mg of macrophage protein (± samplestandard deviation, n = 3).

TABLE 6B Influence of coenzyme Q₁₀ on the uptake of M. mazeiarchaeosomes and conventional liposomes by J774A.1 macrophages as afunction of time, using ³H-chol as a marker.¹ Lipid source for Uptake(cpm)² vesicles 15 min 30 min 45 min 60 min M. mazei + Q₁₀ 5009 ± 61711979 ± 2198 10273 ± 545  51378 ± 6531 DPPC:CHOL + Q₁₀ 1770 ± 340 1385 ±169 1958 ± 232 2589 ± 182 DSPC:CHOL + Q₁₀ 1369 ± 543 1453 ± 239 1599 ±150  2280 ± 230 DSPC:CHOL:DCP + 3450 ± 300 3538 ± 173 2314 ± 3  3241 ±254 Q₁₀ ¹The values were determined by incubating cells with 25 μM ofvesicles for the indicated incubation times. ²cpm of accumulated lipidper mg macrophage protein (± sample standard deviation, n = 3).

TABLE 7A Uptake of M. mazei archaeosomes and conventional liposomes byJ774A.1 macrophages as a function of lipid concentration, using ³H-cholas a marker.¹ Lipid source for Lipid concentration vesicles 25 μM 50 μM75 μM 100 μM M. mazei 3011 ± 642² 14487 ± 3696 7930 ± 773  10140 ± 1412DPPC:CHOL 364 ± 83  1680 ± 572 1332 ± 300  1961 ± 241 DSPC:CHOL 255 ±138 1961 ± 349 1323 ± 205 2049 ± 94 DSPC:CHOL:DCP 365 ± 138 4246 ± 2323147 ± 618 3755 ± 44 ¹The values were determined after incubating cellsfor 60 min with the indicated lipid concentrations. ²Data are shown ascpm of accumulated lipid/mg protein (± sample standard deviation, n =3).

TABLE 7B Influence of coenzyme Q₁₀ on the uptake of M. mazeiarchaeosomes and conventional liposomes by J774A.1 macrophages as afunction of lipid concentration, using ³H-chol as a marker.¹ Lipidsource for Lipid concentration vesicles 25 μM 50 μM 75 μM 100 μM M.mazei + Q₁₀ 27598 ± 190² 44558 ± 1642  32671 ± 7762 23711 ± 3518DPPC:CHOL + Q₁₀ 3497 ± 303 7743 ± 1792 10220 ± 3174 14195 ± 6225DSPC:CHOL + Q₁₀ 2716 ± 691 6499 ± 1400 12389 ± 1311 9226 ± 341DSPC:CHOL:DCP + 3203 ± 425 20411 ± 828  10290 ± 4270 13228 ± 2500 Q₁₀¹The values were determined after incubating cells for 60 min with theindicated vesicle lipid concentrations. ²cpm of accumulated lipid/mgprotein (± sample standard deviation, n = 3).

TABLE 8 Influence of encapsulation of the antigen delivered i.p. on theeffectiveness of various archaeosomes to increase a humoral immuneresponse.¹ Diameter Archaeosome Encapsulation Antibody titreConcentration Carrier/adjuvant (nm) (mg/injection) of Antigen(A_(410 nm)) (μg/ml) M. espanolae 190 ± 61 0.58 Yes 0.536 ± 0.141 41.2M. espanolae 191 ± 60 0.58 No 0.108 ± 0.002 8.3 M. espanolae 191 ± 600.58 No antigen 0.018 ± 0.000 1.4 T. acidophilum 245 ± 94 1.04 Yes 0.385± 0.019 29.6 T. acidophilum 273 ± 84 1.04 No 0.443 ± 0.016 34.1 M.smithii 173 ± 77 2.42 Yes 0.580 ± 0.044 44.6 M. smithii 201 ± 65 2.42 No0.258 ± 0.016 19.9 M. mazei 159 ± 61 1.10 Yes 0.656 ± 0.074 50.5 M.mazei 130 ± 64 1.10 No 0.405 ± 0.054 31.2 M. mazei 130 ± 64 1.10 Noantigen 0.031 ± 0.005 2.4 M. hungatei 218 ± 69 1.2  Yes 0.297 ± 0.05922.9 M. hungatei 287 ± 62 1.2  No 0.342 ± 0.082 26.3 Freund's — None Asemulsion 0.615 ± 0.026 47.3 None — None Bare BSA 0.137 ± 0.002 10.5 ¹25μg of BSA antigen was given per injection i.p. to each mouse.Immunizations where antigen was not encapsulated consisted of BSA in PBSfollowed by bare liposomes 4 hours later. Bare BSA is a control forantigen alone in PBS. Immunizations were given at 0 and 14 days, withblood being taken 4 days following the last boost. ELISA absorbency data(IgG + IgM) are shown for sera diluted 400 fold, and represent theaverage and sample standard deviation for mice in triplicates, with#each serum sample assayed twice. Anti-BSA antibody concentrations arecalculated from the antibody titre data to give μg/ml of undiluted sera.

TABLE 9 Influence of encapsulation of the antigen delivered i.m., oneffectiveness of various archaeosome preparations to increase a humoralimmune response.¹ Archaeosomes Encapsulation Antibody titreCarrier/adjuvant (mg/injection) of Antigen (A_(410 nm)) M. mazei 0.690Yes 0.248 ± 0.056 M. mazei 0.690 No 0.100 ± 0.005 T. acidophilum 1.50 Yes 0.251 ± 0.099 T. acidophilum 1.50  No 0.100 ± 0.033 M. hungatei0.553 Yes 0.107 ± 0.021 M. hungatei 0.553 No 0.071 ± 0.007 Freund's Asemulsion — 0.450 ± 0.026 None Bare BSA — 0.063 ± 0.003 ¹12.5 μg of BSAantigen was given per i.m. injection to the rear haunch. Immunizationswhere antigen was not associated/encapsulated with the archaeosomesconsisted of BSA in PBS injected into one haunch of the mouse and thebare archaeosome injected immediately thereafter into the other haunch.Bare BSA is antigen alone in PBS. Injections were at days 0 and 14, withblood taken 7 days following the last boost. ELISA data (IgG + IgM) areshown for sera diluted 400-fold and represent the #average and samplestandard deviations for mice in triplicates, with each serum sampleassayed twice.

TABLE 10 Influence of injection route on the humoral response to BSAentrapped in M. espanolae archaeosomes.¹ Antibody titre Antibodyconcentration Injection Route (A_(410 nm)) (μg/ml) i.p. 0.773 ± 0.109118.9 s.c. 0.493 ± 0.065 75.8 i.m. 0.535 ± 0.066 82.3 ¹BSA entrapped inM. espanolae archaeosomes of 147 ± 59 nm diameter (25 μg BSA in 0.24 mglipid/injection) was used to inject mice at days 0, and 14. Blood wastaken on day 18. Antibody titres (IgG + IgM) are shown as the averageand sample standard deviation for 3 mice, with each serum assayed induplicate. Absorbency data are shown for sera diluted 800 fold; anti-BSAantibody concentrations are calculated as μg/ml of undiluted sera.

TABLE 11 Relationship between the percentage of archaeobacterial lipidsused in the preparation of vesicles and their ability to serve as acarrier/adjuvant for an entrapped antigen.¹ Immune archaeobacterialDiameter Vesicles/ response Carried/Adjuvant lipids (%) (nm) injection(mg) (Relative %) Freund's none — — 100 ± 7.0  None none — — 6.9 ± 0.3Archaeosomes 100  180 ± 81 1.19 91.4 ± 5.9  Archaeosomes 50 186 ± 570.86 44.0 ± 11.5 Archaeosomes 10 152 ± 54 1.14 22.4 ± 3.9  Liposomes  0126 ± 56 1.01 9.8 ± 0.9 ¹Mice (3 per group) were immunized by i.p.injections of various adjuvants containing 12.5 μg of BSA, as antigen.Vesicles were prepared from different ratios by weight of TPL fromMethanobrevibacter smithii (archaeobacterial lipids) and DMPC:DMPG. Thedry weight of vesicles given per injection was calculated to deliver aconstant amount of 12.5 μg antigen. Mice were immunized at days 0 and14. Blood was taken at day 20, and the immune response determined astitres of IgG + #IgM antibodies in the serum using the ELISA method.

TABLE 12 Humoral response to a protein antigen entrapped in archaeosomesprepared from purified archaeal lipids.¹ Adjuvants/ Diameter (nm)²Antibody titres (A_(410 nm)) Carrier Day 0 Day 14 Day 25 Day 35 Day 55None — — 0.064 ± 0.001 0.082 ± 0.007 0.075 ± 0.008 Freund's — — 0.610 ±0.072 0.606 ± 0.069 0.385 ± 0.066 PGP-Me 124 ± 54 137 ± 52 0.574 ± 0.0900.540 ± 0.124 0.290 ± 0.088 D_(OH)PI 140 ± 59 138 ± 58 0.232 ± 0.0220.238 ± 0.051 0.143 ± 0.032 D_(OH)PG 217 ± 71 192 ± 75 0.401 ± 0.1070.332 ± 0.044 0.199 ± 0.020 Tetraether  229 ± 100  202 ± 103 0.338 ±0.029 0.238 ± 0.028 0.178 ± 0.004 ¹12.5 μg BSA was injected i.p. permouse at days 0 and 14. Blood was taken from the tail vein at the timesindicated from first injection (25-55 days). Antibody absorbency dataare shown for sera diluted by a factor of 400, and represent the averageand sample standard deviation for triplicate mice, with each serumassayed twice. ²Vesicle diameters were determined at the time of thefirst (day 0) and second (day 14) injections.

We claim:
 1. A liposome composition comprising (1) the total polarlipids extract of an archaeobacterium, (2) a pharmaceutical agent and(3) coenzyme Q₁₀.
 2. A liposome composition according to claim 1,further comprising (4) a polyethyleneglycol lipid conjugate.
 3. Aliposome composition according to claim 1, wherein the archaeobacteriumis selected from the group consisting of Methanosarcina mazei,Methanospirillum hungatei, Methanosphaera stadtmanae, Methanobrevibactersmithii, Methanococcus voltae, Methanobacterium espanolae, Methanosaetaconcilii, and Thermoplasma acidophilum.
 4. A liposome compositionaccording to claim 1, wherein the liposome is unilamellar.
 5. A liposomecomposition according to claim 1, wherein the liposome size is in therange of not less than 50 nm, but less than 500 nm, in diameter.
 6. Aliposome composition according to claim 1 wherein the archaeobacteriumis Methanosarcina mazei.
 7. A liposome composition according to claim 1,wherein the archaeobacterium is Thermoplasma acidophilum.
 8. A methodfor the delivery of a pharmaceutical agent to an animal, comprisingadministering to the animal a liposome prepared from a compositionconsisting essentially of the total polar lipids extract of anarchaeobacterium and coenzyme Q₁₀, as a carrier for said pharmaceuticalagent.
 9. A method for the delivery of a pharmaceutical agent to ananimal, comprising administering to the animal a liposome prepared froma composition consisting essentially of the total polar lipids extractof an archaeobacterium, coenzyme Q₁₀, and a polyethyleneglycol lipidconjugate, as a carrier for said pharmaceutical agent.
 10. A methodaccording to claim 9, wherein the liposome is administered to an animalorally, intraperitoneally, intramuscularly, subcutaneously, orintravenously.
 11. A method according to claim 9, herein thearchaeobacterium is Methanosarcina mazei.
 12. A method for enhancing thetargeted delivery of a pharmaceutical agent to specific animal organs,comprising administering to the animal a liposome prepared from acomposition consisting essentially of the total polar lipids extract ofan archaeobacterium, coenzyme Q₁₀, and a polyethyleneglycol lipidconjugate, as a carrier for said pharmaceutical agent.
 13. A methodaccording to claim 12,wherein the liposome is delivered to an animal viathe oral route.
 14. A method for the delivery of a pharmaceutical orbiological agent to phagocytic cells of an animal, comprisingadministering to the animal a liposome prepared from a compositionconsisting essentially of the total polar lipids extract of anarchacobacterium as a carrier for said pharmaceutical or biologicalagent.
 15. A method according to claim 14, wherein the pharmaceutical orbiological agent is selected from an antibiotic, antiviral agent,vitamin, imaging agent, enzyme, DNA or hormone.
 16. A method oftreatment for a disease caused by a pathogen residing inside the cellsof an animal, comprising administering to the animal a liposome preparedfrom the total polar lipids extract of an archaeobacterium and anantimicrobial or an antiviral agent.
 17. A method of imaging of tissuesand organs in an animal, comprising administering to the animal aliposome prepared from a composition consisting essentially of the totalpolar lipids extract of an archaeobacterium as a carrier for an imagingagent.
 18. A method for the selective delivery of a pharmaceutical orbiological agent to specific tissues of an animal, comprisingadministering to the animal a liposome prepared from a compositionconsisting essentially of the total polar lipids extract of anarchaeobacterium and as a carrier for said pharmaceutical or biologicalagent.
 19. A method according to claim 18, wherein the pharmaceutical orbiological agent is selected from an antibiotic, antiviral agent,vitamin, imaging agent, enzyme, DNA or hormone.
 20. A method accordingto claim 18, wherein the liposome composition additionally comprises apolyethyleneglycol lipid conjugate.
 21. A method according to any one ofclaims 14, 15, 16, 17, 18, 19 or 20, wherein the archaeobacterium isselected from the group consisting of Methanosarcina mazei,Methanospirillum hungatei, Methanosphaera stadtmanae, Methanobrevibactersmithii, Methanococcus voltae, Methanobacterium espanolae, Methanosaetaconcilii and Thermoplasma acidophilum.
 22. A method according to any oneof claims 14, 15, 16, 17, 18, 19 or 20, wherein the dosage of liposomesto be delivered is 4 to 73 mg/kg of animal body weight.
 23. A methodaccording to any one of claims 14, 15, 16, 17, 18, 19 or 20, wherein thearchaeobacterium is Methanosarcina mazei.
 24. A method according to anyone of claim 14, 15, 16, 17, 18, 19 or 20, wherein the liposome isadministered to an animal orally, intraperitoneally, intramuscularly,subcutaneously, or intravenously.